The present invention relates generally to polyketides and polyketide synthases. In particular, the invention pertains to novel methods of producing polyketides and libraries of polyketides using a cell-free system.
Polyketides are a large, structurally diverse family of natural products. Polyketides possess a broad range of biological activities including antibiotic and pharmacological properties. For example, polyketides are represented by such antibiotics as tetracyclines and erythromycin, anticancer agents including daunomycin, immunosuppressants, for example FK506 and rapamycin, and veterinary products such as monensin and avermectin.
Polyketides occur in most groups of organisms and are especially abundant in a class of mycelial bacteria, the actinomycetes, which produce various polyketides. Polyketide synthases (PKSs) are multifunctional enzymes related to fatty acid synthases (FASs). PKSs catalyze the biosynthesis of polyketides through repeated (decarboxylative) Claisen condensations between acylthioesters, usually acetyl, propionyl, malonyl or methylmalonyl. Following each condensation, they introduce structural variability into the product by catalyzing all, part, or none of a reductive cycle comprising a ketoreduction, dehydration, and enoylreduction on the xcex2-keto group of the growing polyketide chain. PKSs incorporate enormous structural diversity into their products, in addition to varying the condensation cycle, by controlling the overall chain length, choice of primer and extender units and, particularly in the case of aromatic polyketides, regiospecific cyclizations of the nascent polyketide chain. After the carbon chain has grown to a length characteristic of each specific product, it is released from the synthase by thiolysis or acyltransfer. Thus, PKSs consist of families of enzymes which work together to produce a given polyketide. It is the controlled variation in chain length, choice of chain-building units, and the reductive cycle, genetically programmed into each PKS, that contributes to the variation seen among naturally occurring polyketides.
Two general classes of PKSs exist. These classifications are well known. See, for example, Hopwood, D. A. and Khosla, C., Secondary Metabolites: Their Function and Evolution (1992) Wiley Chichester (Ciba Foundation Symposium 171) pp. 88-112.
One class, known as Type I or modular PKSs, is represented by the PKSs which catalyze the biosynthesis of complex polyketides such as erythromycin and avermectin. These xe2x80x9cmodularxe2x80x9d PKSs include assemblies of several large multifunctional proteins carrying, between them, a set of separate active sites for each step of carbon chain assembly and modification (Cortes, J. et al. Nature (1990) 348:176; Donadio, S. et al. Science (1991) 252:675; MacNeil, D. J. et al. Gene (1992) 115:119). The active sites required for one cycle of condensation and reduction are clustered as xe2x80x9cmodulesxe2x80x9d (Donadio et al. Science (1991), supra; Donadio, S. et al. Gene (1992) 111:51). For example, 6-deoxyerythronolide B synthase (DEBS) consists of the three multifunctional proteins, DEBS 1, DEBS 2, and DEBS 3 (Caffrey, P. et al. FEBS Letters (192) 304:225), each of which possesses two modules. (See FIG. 1.)
As described below, a module contains at least the minimal activities required for the condensation of an extender unit onto a growing polyketide chain; the minimal activities required are a ketosynthase (KS), an acyl transferase (AT) and an acyl carrier protein (ACP). Additional activities for further modification reactions such as a reductive cycle or cyclization may also be included in a module. Structural diversity occurs in this class of PKSs from variations in the number and type of active sites in the PKSS. This class of PKSs displays a one-to-one correlation between the number and clustering of active sites in the primary sequence of the PKS and the structure of the polyketide backbone.
The second class of PKSs, the aromatic or Type II PKSs, has a single set of iteratively used active sites (Bibb, M. J. et al. EMBO J. (1989) 8:2727; Sherman, D. H. et al. EMBO J. (1989) 8:2717; Fernandez-Moreno, M. A. et al. J. Biol. Chem. (1992) 267:19278). Streptomyces is an actinomycete which is an abundant producer of aromatic polyketides. In each Streptomyces aromatic PKS so far studied, carbon chain assembly requires the products of three open reading frames (ORFs). (See FIG. 2.) ORF1 encodes a ketosynthase (KS) and an acyltransferase (AT) active site (KS/AT); ORF2 encodes a chain length determining factor (CLF), a protein similar to the ORF1 product but lacking the KS and AT motifs; and ORF3 encodes a discrete acyl carrier protein (ACP). Some gene clusters also code for a ketoreductase (KR) and a cyclase, involved in cyclization of the nascent polyketide backbone. However, it has been found that only the KS/AT, CLF, and ACP, need be present in order to produce an identifiable polyketide.
Fungal PKSs, such as the 6-methylsalicylic acid PKS, consist of a single multidomain polypeptide which includes all the active sites required for the biosynthesis of 6-methylsalicylic acid (Beck, J. et al. Eur. J. Biochem. (1990) 192:487-498; Davis, R. et al. Abstr. of the Genetics of Industrial Microorganism Meeting, Montreal, abstr. P288 (1994)). Fungal PKSs incorporate features of both modular and aromatic PKSs.
Streptomyces coelicolor produces the blue-pigmented polyketide, actinorhodin. The actinorhodin gene cluster (act), has been cloned (Malpartida, F. and Hopwood, D. A. Nature (1984) 309:462; Malpartida, F. and Hopwood, D. A. Mol. Gen. Genet. (1986) 205:66) and completely sequenced (Fernandez-Moreno et al. J. Biol. Chem. (1992), supra; Hallam, S. E. et al. Gene (1988) 74:305; Fernandez-Moreno, M. A. et al. Cell (1991) 66:769; Caballero, J. et al. Mol. Gen. Genet. (1991) 230:401). The cluster encodes the PKS enzymes described above, a cyclase and a series of tailoring enzymes involved in subsequent modification reactions leading to actinorhodin, as well as proteins involved in export of the antibiotic and at least one protein that specifically activates transcription of the gene cluster. Other genes required for global regulation of antibiotic biosynthesis, as well as for the supply of starter (acetyl-CoA) and extender (malonyl-CoA) units for polyketide biosynthesis, are located elsewhere in the genome.
The act gene cluster from S. coelicolor has been used to produce actinorhodin in S. parvulus. Malpartida, F. and Hopwood, D. A. Nature (1984) 309:462.
Bartel et al. J. Bacteriol. (1990) 172:4816-4826, recombinantly produced aloesaponarin II using S. galilaeus transformed with an S. coelicolor act gene cluster consisting of four genetic loci, acti, actIII, actIV and actVII. Hybrid PKSs, including the basic act gene set but with ACP genes derived from granaticin, oxytetracycline, tetracenomycin and frenolicin PKSs, have also been designed which are able to express functional synthases. Khosla, C. et al. J. Bacteriol. (1993) 175:2197-2204. Hopwood, D. A. et al. Nature (1985) 314:642-644, describes the production of hybrid aromatic polyketides, using recombinant techniques. Sherman, D. H. et al. J. Bacteriol. (1992) 174:6184-6190, reports the transformation of various S. coelicolor mutants, lacking different components of the act PKS gene cluster, with the corresponding granaticin (gra) genes from S. violaceoruber, in trans.
Although the above described model for complex polyketide biosynthesis by modular (Type I) PKSs has been substantiated by radioisotope and stable isotope labeling experiments, heterologous expression, directed mutagenesis, and in vitro studies on partially active proteins, cell-free enzymatic synthesis of complex polyketides has proved unsuccessful despite more than 30 years of intense efforts (Caffrey et al. FEBS Letters (1992), supra; Aparicio, J. F. et al. J. Biol. Chem. (1994) 269:8524; Bevitt, D. J. et al. Eur. J. Biochem. (1992) 204:39; Caffrey, P. et al. Eur. J. Biochem. (1991) 195:823); Leadlay, P. F. et al. Biochemem. Soc. Trans. (1993) 21:218; Marsden, A. F. A. et al. Science (1994) 263:378; Wawszkiewicz, E. J. et al. Biochememische Z. (1964) 340:213; Corcoran, J. W. et al. in Proc. 5th Int. Congr. Chemother. (Vienna, 1967), Abstracts of Communications, p. 35; Corcoran, J. W. et al. in Antibiotics IV. Biosynthesis (1982) Corcoran, J. W., Ed. (Springer-Verlag, New York) p. 146; Roberts, G. FEBS Lett. (1983) 159:13; Roberts, G. et al. Biochemical Soc. Trans. (1984) 12:642; Hunaiti, A. A. et al. Antimicrob. Agents. Chemother. (1984) 25:173). This is due, in part, to the difficulty of isolating fully active forms of these large, poorly expressed multifunctional proteins from naturally occurring producer organisms and, in part, to the relative lability of intermediates formed during the course of polyketide biosynthesis. For example, the three DEBS proteins have been purified individually from the natural producer organism, Saccharopolyspora erythraea (Caffrey et al. FEBS Letters (1992), supra; Aparicio et al. J. Biol. Chem. (1994), supra; Bevitt et al. Eur. J. Biochem. (1992), supra; Caffrey et al. Eur. J. Biochem. (1991), supra; Leadlay et al. Biochem. Soc. Trans. (1993); Marsden et al. Science (1994), supra). Studies on the purified enzymes facilitated clarification of their stereospecificity, showing that 2S-methylmalonyl-CoA is the extender substrate for all 6 acyltransferase sites (Marsden et al. Science (1994), supra), thereby implying that the differing configurations of the methyl-branched centers result from selective epimerization of specific enzyme-bound intermediates. However, the lack of a full turnover assay prevented these investigators from probing the mechanisms of the enzyme complex in greater detail.
In an attempt to overcome some of these limitations, modular PKS subunits have been expressed in heterologous hosts such as E. coli (Aparicio et al. J. Biol. Chem. (1994), supra; Bevitt et al. Eur. J. Biochem. (1992), supra; Caffrey et al. Eur. J. Biochem. (1991), supra; Leadlay et al. Biochem. Soc. Trans. (1993);) and S. coelicolor (Kao, C. M. et al. Science (1994) 265:509; International Publication No. WO 95/08548 (published Mar. 30, 1995)). Whereas the proteins expressed in E. coli are not fully active, heterologous expression in S. coelicolor resulted in production of active protein as demonstrated by the production of 6-deoxyerythronolide (xe2x80x9c6-DEBxe2x80x9d) in vivo. Cell-free enzymatic synthesis of polyketides from simpler PKSs such as the 6-methylsalicylate synthase (Dimroth, P. et al. Eur. J. Biochem. (1970) 13:98; Beck, J. et al. Eur. J. Biochem. (1990) 192:487); Spencer J. B. et al. Biochem. J. (1992) 288:839), chalcone synthase (Lanz, T. et al. J. Biol. Chem. (1991) 266:9971 (1991)), and the tetracenomycin synthase (Shen, B. et al. Science (1993) 262:1535) has been reported.
However, no one to date has described the cell-free enzymatic synthesis of polyketides from modular PKSs, or has used a cell-free system to produce libraries containing a multiplicity of different polyketides.
The present invention provides methods to produce both novel and known polyketides. In one embodiment, a cell-free system comprising a modular PKS effects synthesis of a polyketide when incubated with an appropriate substrate set.
In another embodiment, the invention is directed to a method of synthesizing a library containing a multiplicity of different polyketides by use of cell-free systems and to a matrix of cell-free subsystems for the production of these libraries.
Thus, in one aspect, the invention is directed to a method comprising providing one or more proteins comprising at least two modules of a modular polyketide synthase in a cell-free system; adding to said system at least one starter unit and at least one extender unit; incubating said cell-free system containing said starter unit and extender unit under conditions wherein said polyketide is synthesized; and optionally recovering the polyketide from the cell-free system.
In another aspect, the invention is directed to a matrix for the production of a polyketide library which comprises a series of cell-free subsystems each containing one or more polyketide synthase proteins comprising enzymatic activities that effect the coupling of at least one extender unit to a starter unit, including a growing polyketide chain; each said subsystem containing at least one starter unit and at least one extender unit; and wherein at least one enzymatic activity or at least one extender unit or at least one starter unit or is different as between each subsystem.
The invention in another aspect is directed to methods to prepare libraries of polyketides using these matrices.
In yet another aspect, the invention is directed to method to produce a desired polyketide which method comprises: providing a system comprising a functional modular polyketide synthase (PKS), or a functional portion thereof, wherein said PKS cannot be loaded with a natural first-module starter unit, or wherein, once loaded, cannot catalyze the condensation of an extender unit to the first-module starter unit to produce a polyketide intermediate; adding to said system a starter unit that is a substrate for the PKS; incubating the system containing said PKS and said starter unit substrate under conditions wherein said polyketide is synthesized; and optionally recovering the polyketide.
In still another aspect, the invention is directed to a functional modular polyketide synthase system, or a functional portion thereof, which cannot be loaded with a natural first-module starter unit, or which, once loaded, cannot catalyze the condensation of an extender unit to the first-module starter unit to produce a polyketide intermediate.